Dissociated xenograft tumor single-cell suspensions were thawed in at least 5X dilution volume of appropriate culture media and resuspended in PBS with 2% FBS and 2mM EDTA. Viable cells were isolated by FACS sorting as described above and scRNA-seq, downstream library preparation, and sequencing was performed in the same manner as patient samples with the exception of the following: raw sequencing data was mapped to the combined mm10 and hg19 reference genomes to identify murine and cancer cell transcripts, respectively, with the 10X cellranger pipeline (version 2.1.0). For in vitro scRNA-seq, cultured CAL27 cells were washed with PBS twice after trypsinization, placed in PBS with 0.04% BSA, counted and directly loaded onto a 10X Chromium controller. Library preparation was performed with the Single Cell 3′ v3 kit (10X Genomics) per manufacturer’s protocol. Sequencing was performed on an Illumina HiSeq 4000 at a depth of 52,740 mean reads/cell. Raw sequencing data was mapped to the GrCH38 transcriptome with 10X cellranger version 3.0.2.
Single cell 3 v3 kit
Manufactured by 10x Genomics
The Single Cell 3' v3 kit is a laboratory equipment product from 10x Genomics. It is designed for single-cell gene expression analysis, enabling researchers to study the transcriptomes of individual cells.
Automatically generated - may contain errors
2 protocols using single cell 3 v3 kit
1
Xenograft Tumor Single-Cell Sequencing
2
Blastoid Single-Cell RNA-seq Workflow
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Single cells were obtained upon enzymatic dissociation from day 7 and day 10 (Geltrex 5%) in vitro attached blastoids. Two different methods were used for sample preparation according to the presence of extracellular matrix in the medium. For day 7 blastoids, treatment with Accutase (STEMCELL) for 10 min at 37°C was sufficient to detach and isolate a single-cell suspension. To remove the extracellular matrix from the day-10 sample, we first applied Dispase solution (STEMCELL) for 7 min, washed it twice with PBS−/−, and applied Papain/DNase solution (Worthington) for 15 min at 37°C. Mechanical trituration followed by two washes with 0.04% BSA in PBS−/− was used to isolate single cells. Before Gem formation, samples were filtered with a 40-μm strainer. A Single Cell 3′ v.3 kit (10× Genomics) was used for library preparation according to the manufacturer’s instructions.
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